Lab 7: Biochemical Testing for Microbial Identification - Methyl Red, Voges-Proskauer, and Catalase Testing

Identifies metabolic properties of microbes

biochemical test

Enzyme that mediates the breakdown of hydrogen peroxide

catalase

Anaerobic metabolism that produces acids, alcohol, and gases

mixed acid fermentation

Anaerobic metabolism that produces acetoin

butanediol fermentation

Metabolic process resulting in oxygen and peroxide by-products

aerobic respiration

What 2 terms apply to voges-Proskauer testing?

Aerobic respiration

Mixed-Acid fermentation

peroxide

Barritt's reagents

Low pH

Butanediol fermentation

Butanediol fermentation

and

Barritt's reagents

What 2 terms apply to Methyl red testing?

Aerobic respiration

Mixed-Acid fermentation

peroxide

Barritt's reagents

Low pH

Butanediol fermentation

Low pH

and

Mixed-Acid fermentation

What 2 terms apply to catalase testing?

Aerobic respiration

Mixed-Acid fermentation

peroxide

Barritt's reagents

Low pH

Butanediol fermentation

Aerobic respiration

and

peroxide

T/F: MR-VP broth contains nutrients and buffers

True

T/F: Fermentation occurs when carbohydrates are metabolized anaerobically

True

an anaerobic metabolic process that produces lactic acid, acetic acid, ethanol, carbon dioxide and hydrogen gas.

Mixed-acid fermentation

What do the products of mixed acid fermentation do to the pH do the buffered media?

Lower the pH

pH indicator that turns red at acidic pHs between 4.4 and 6.0.

methyl red

Cultures demonstrating mixed-acid fermentation turn _______ when methyl red is added to the broth

red

Why does the cultures turn red when metyl red is added?

because the mixed-acid fermentation creates acidic by-products that do not neutralize in the buffered solution

Cultures that do not undergo mixed-acid fermentation turn ______ when methyl red is added, indicating a negative test result.

yellow

T/F: All aerobic microbes lack the enzyme catalse

False

test is designed to identify microbes that ferment glucose via the butanediol pathway.

Voges-Proskauer test

What test are Barritt's Reagents used for?

Voges-Proskauer test

Butanediol fermentation produces _______ acids than mixed-acid fermentation.

weaker

The intermediate of butanediol fermentation

acetoin

acetoin turns _____ in the presence of Barritts reagent

red

What does red result mean for Voges-Proskauer test?

positive for butanediol pathway.

Why are the methyl red and Voges-Proskauer tests are typically performed together?

to differentiate bacteria within the family Enterobacteriaceae.

T/F: Biochemical tests provide more generalized information than do morphological observations

False

Biochemical tests are designed to identify _____ properties of microbes.

morphologic

metabolic

motility

All of the above

metabolic

Mixed-acid fermentation produces _____.

acetic acid

ethanol

hydrogen gas

All of the above

All of the above

Methyl red is a pH indicator that turns red in _____ conditions.

acidic

T/F: Butanediol fermentation produces weaker acids than mixed-acid fermentation.

True

Bacteria that live in oxygenated environments produce the enzyme _____ to neutralize the harmful effects of hydrogen peroxide.

catalase

______ microbes generally lack the catalase enzyme.

Anaerobic

Enzyme that

mediates the breakdown of hydrogen peroxide into oxygen and water.

What does positive catalase test mean? (BUbbles form)

aerobic microbes

What does negative catalase test mean?

anaerobic microbe

Is E coli catalase positive or negative?

catalase positive (ANAEROBIC)

Is S. epidermidis catalase positive or negative?

catalase positive (ANAEROBIC)

WHat does facultative anaerobe mean?

can respire aerobically or anaerobically

What does obligate anaerobe mean?

requires absence of oxygen, CANNOT BE AROUND O2

Is E coli MR + or -

MR +

Is E coli positive or negative for Voges Proskauer test?

VP -

Is S. epi MR + or -

MR - (It does NOT do mixed acid fermentation)

Is S. epi VP + or -

VP + (It goes go through the butanediol pathway, producing acetoin)

Catalase neutralizes the harmful effects of _____.

hydrogen peroxide

butanediol

acetoin

None of the above

hydrogen peroxide

Why are biochemical tests used to identify microbes?

Biochemical tests are used to identify microbes because they allow us to identify metabolic properties of different bacteria, which can lead us to identify the organism. These tests are also more specific than just morphological observations

What is mixed-acid fermentation? How is methyl red used to determine if a microbe has undergone mixed-acid fermentation?

Mixed-acid fermentation is an anaerobic process that produces lactic acid, acetic acid, ethanol, carbon dioxide, and hydrogen gas. These products lower the pH of the media that the microbes are growing in, which can allow for methyl red to turn red when placed in the acidic media. If the solution turns red, than this is a positive sign that a microbe has undergone mixed-acid fermentation. If the solution turns yellow, this is a negative test result.

Why are the methyl red and Voges-Proskauer tests often performed together?

Methyl red and Voges-Proskauer tests are done together because they can differentiate bacteria within the family Enterobacteriaceae.

How do the experimental results relate to the metabolic pathways used by E. coli and S. epidermidis?

E. coli goes would have a positive methyl red test since it goes through mixed acid fermentation, and a negative Voges-Proskauer test since it does not go through the butanediol pathway.

S. epidermidis would be negative for methyl red (turns yellow), and positive for Voges-Proskauer.

What is catalase? What types of microbes are more likely to produce catalase?

Catalase is an enzyme that mediates the breakdown of hydrogen peroxide into oxygen and water. Microbes that are aerobic are more likely to produce catalase.

Based on the results from your experiment, what can you conclude about the metabolism of E. coli and S. epidermidis?

Since bubbles formed, this means that E. coli and S. epidermidis is positive for catalase. This overall means that both these microbes are aerobic and have catalase to neutralize the harmful effects of hydrogen peroxide.

The _____ is a biochemical test used to determine the identity of an unknown microbe.

methyl red test

Voges-Proskauer test

catalase test

All of the above

All of the above

T/F: Fermentation occurs when carbohydrates are broken down aerobically.

False

*Fermentation occurs when carbohydrates are broken down anaerobically.

The methyl red test detects a microbe's ability to _____.

perform mixed-acid fermentation

perform butanediol fermentation

respire aerobically

All of the above

perform mixed-acid fermentation

The products of mixed-acid fermentation _____ the pH of buffered media.

lower

raise

neutralize

None of the above

lower

Butanediol fermentation produces _____ acids compared to mixed-acid fermentation.

stronger

weaker

identical

None of the above

weaker

T/F: The methyl red and Voges-Proskauer tests are typically performed together to differentiate bacteria within the family Enterobacteriaceae.

True

Oxygen ions and _____ are metabolites of aerobic respiration that can damage cell structures.

alcohols

peroxides

mixed acids

None of the above

peroxides

____ broth is used to culture microbes for the methyl red and Voges-Proskauer​ tests.

MR-VP

Phenol red

Nutrient

None of the above

MR-VP

Red-stained media is a _____ result for the methyl red test.

positive

negative

inconclusive

positive

A positive Voges-Proskauer test appears as _____.

solid red media

solid yellow media

a red layer atop yellow media

None of the above

a red layer atop yellow media

_____ is a positive result for catalase testing.

Bubbling peroxide

Red media

Yellow media

None of the above

Bubbling peroxide

A microbe produced positive methyl red results and negative Voges-Proskauer results. It can be concluded that the microbe _____.

respires aerobically

produces mixed acids but does not use the butanediol pathway

uses the butanediol pathway but does not produce mixed acids

None of the above

produces mixed acids but does not use the butanediol pathway

What is the oxidase test?

test is used to detect the presence of the enzyme cytochrome oxidase which is used for the reduction of oxygen at the end of the electron transport chain. A positive test means that the sample has the cytochrome oxidase enzyme and the sample turns blue. A negative test means that the sample doesn't have the cytochrome oxidase enzyme, and it does not change color.

What are some oxidase positive microbes?

many gram-negative bacteria, Pseudomonas aeruginosa, and Pasteurella multocida

What are some oxidase negative microbes?

E. coli, S. epidermidis, and Enterobacteriaceae